One of the most important challenges facing analytical chemistry today is analysis of biological samples. A successful technique should handle an immense number of samples in a short time and be compatible with existing liquid-phase chemical and separation techniques.
One of the most useful analytical methods for biological samples is mass spectroscopy. Liquid samples can be introduced into a mass spectrometer by electrospray ionization (1), a process that creates multiple charged ions. However, multiple ions can result in complex spectra and reduced sensitivity. A more preferred technique, matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) (2), has received prominence in analysis of biological polymers for its excellent characteristics, such as ease of sample preparation, predominance of singly charged ions in mass spectra, sensitivity and high speed. In principle, a mixture of analytes with excess matrix is deposited onto a probe and irradiated by a short laser pulse. Matrix molecules, which absorb most of the laser energy, transfer that energy to analyte molecules to vaporize and ionize them. Once created, the analyte ions are analyzed in mass spectrometer, typically a TOF mass spectrometer.
MALDI is typically operated as an off-line ionization technique, where the sample, mixed with a suitable matrix, is deposited on the MALDI target to form dry mixed crystals and, subsequently, placed in the source chamber of the mass spectrometer. Although solid samples provide excellent results, the sample preparation and introduction into the vacuum chamber requires a significant amount of time. Even simultaneous introduction of several solid samples into a mass spectrometer or off-line coupling of liquid-phase separation techniques with a mass spectrometer do not use TOF mass spectrometer time efficiently. In addition, MALDI-MS analysis typically requires finding the "sweet spot" on the sample target, so that a reasonable signal can be obtained (5,6). Although a motorized x-y stage may be incorporated for automated searching for the spot providing the best spectrum, this procedure can be a time consuming step.
To improve on these procedures, microfabricated targets have recently been developed for automated high throughput MALDI analysis (7,8). In these designs, pL--nL sample volumes can be deposited into a microfabricated well with dimensions similar to the spot size of the desorbing laser beam (.about.100 .mu.m diameter). Thus, the whole sample spot can be irradiated and the search for the "sweet spot" eliminated. Analysis of short oligonucleotides has been demonstrated with .about.3.3 s required to obtain a good signal to noise ratio for each sample spot (8). Although the total analysis time, including the data storage, required 43 min, theoretically all 96 samples could be recorded in 330s.
While the miniaturization of the sample target simplifies the static MALDI analysis, on-line coupling would allow continuous analysis of liquid samples including direct sample infusion and the monitoring of chromatographic and electrophoretic separations. Compared to ESI, MALDI provides less complex spectra and, potentially, higher sensitivity. There have been numerous reports in the literature about the MALDI analysis of flowing liquid samples. In one arrangement, the sample components exiting a CE separation capillary were continuously deposited on a membrane presoaked with the matrix and analyzed after drying (9,10,11,12). In other cases, the liquid samples were analyzed directly inside the mass spectrometer using a variety of matrices and interfaces. For example, a nebulizer interface was used for continuous sample and matrix introduction (13-19). MALDI was then performed directly off rapidly dried droplets. In another design, a continuous probe, similar to a fast atom bombardment (FAB) (20) interface, was used for the analysis of a flowing sample stream with liquid matrix (21-24). Glycerol was used to prevent freezing of the sample. Other attempts for liquid sample desorption were also made using fine dispersions of graphite particles (25, 26,27) and liquid matrices (2,28-40) instead of a more conventional matrices. More recently, an outlet of the capillary electrophoresis column was placed directly in the vacuum region of the TOF mass spectrometer (41). The sample ions, eluting in a solution of CuCl.sub.2, were desorbed by a laser irradiating the capillary end. On line spectra of short peptides separated by CE were recorded. Attempts to use ESI to introduce liquid sample directly to the evacuated source of a mass spectrometer have also been reported (42-44).
Although the above-listed examples show efforts to address various different problems related to the analysis of flowing liquid samples, currently there is no universal MALDI interface that would address the need for simple and sensitive analysis of minute sample amounts and, furthermore, that would permit simultaneous, on-line processing of multiple samples. A generally useful procedure and a universal interface for continuous introduction of an individual or multiplexed liquid sample or samples into a time-of-flight mass spectrometer so that on-line MALDI-MS analysis can be carried out would be highly desirable.